U.S. patent application number 10/706391 was filed with the patent office on 2004-07-15 for anti-microbial targeting chimeric pharmaceutical.
Invention is credited to Anderson, Maxwell H., Eckert, Randal, Qi, Fengxia, Shi, Wenyuan.
Application Number | 20040137482 10/706391 |
Document ID | / |
Family ID | 46300323 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137482 |
Kind Code |
A1 |
Eckert, Randal ; et
al. |
July 15, 2004 |
Anti-microbial targeting chimeric pharmaceutical
Abstract
The present invention is based on the discovery of a composition
that provides targeted anti-microbial effect. Specifically the
composition contains a targeting moiety which recognizes a target
microbial organism and an anti-microbial peptide moiety which has
anti-microbial activity. In addition, the present invention
provides methods of treating a microbial infection, e.g., on
mucosal surfaces by using the compositions provided by the present
invention.
Inventors: |
Eckert, Randal; (Los
Angeles, CA) ; Qi, Fengxia; (Harbor City, CA)
; Shi, Wenyuan; (Los Angeles, CA) ; Anderson,
Maxwell H.; (Seattle, WA) |
Correspondence
Address: |
GRAY CARY WARE & FREIDENRICH LLP
153 TOWNSEND
SUITE 800
SAN FRANCISCO
CA
94107
US
|
Family ID: |
46300323 |
Appl. No.: |
10/706391 |
Filed: |
November 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10706391 |
Nov 12, 2003 |
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10077624 |
Feb 14, 2002 |
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10077624 |
Feb 14, 2002 |
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09910358 |
Jul 19, 2001 |
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09910358 |
Jul 19, 2001 |
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09378577 |
Aug 20, 1999 |
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Current U.S.
Class: |
435/6.15 |
Current CPC
Class: |
C07K 2317/24 20130101;
A61K 47/6811 20170801; C07K 2317/21 20130101; A61K 47/6835
20170801; A61K 2039/505 20130101; C07K 2317/50 20130101; C07K
2319/00 20130101; Y02A 50/403 20180101; Y02A 50/30 20180101; A61K
47/6809 20170801; A61K 47/6875 20170801; C07K 16/1275 20130101;
A61P 31/00 20180101; C12N 15/8258 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A composition useful for treatment of microbial organisms
comprising a targeting moiety and an anti-microbial peptide moiety,
wherein the targeting moiety is coupled to the anti-microbial
peptide moiety and recognizes a target microbial organism and
wherein the composition has an anti-microbial effect on the target
microbial organism.
2. The composition of claim 1, wherein the targeting moiety is a
peptide.
3. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO. 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, or 61.
4. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO. 24,
25, 26, 27, 28, 29, 30, 31, 32, or 33 and wherein the target
microbial organism is Pseudomonas.
5. The composition of claim 1, wherein the target microbial
organism is P. aeroginosa.
6. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO. 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51
and wherein the target microbial organism is Staphylococcus.
7. The composition of claim 6, wherein the target microbial
organism is S. aureus.
8. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO. 52,
53, 54, 55, 56, 57, 58, 59, or 60 and wherein the target microbial
organism is E. coli.
9. The composition of claim 8, wherein the target microbial
organism is E. coli DH5.alpha..
10. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO.
61.
11. The composition of claim 10, wherein the target microbial
organism is Pseudomonas.
12. The composition of claim 10, wherein the target microbial
organism is E coli.
13. The composition of claim 10, wherein the targeting moiety is
coupled to the C terminus of the anti-microbial peptide moiety.
14. The composition of claim 1, wherein the targeting moiety is a
peptide having an amino acid sequence as shown in SEQ ID NO. 61 and
the anti-microbial peptide moiety is novispirin G10 having an amino
acid sequence as shown in SEQ ID NO. 16.
15. The composition of claim 14, wherein the targeting moiety is
coupled to the C terminus of novispirin G10.
16. The composition of claim 14, wherein the targeting moiety and
the anti-microbial peptide moiety are fused via a peptide linker to
form a fusion peptide and wherein the fusion peptide comprises an
amino acid as shown in SEQ ID NO. 70.
17. The composition of claim 16, wherein the fusion peptide
comprises an amino acid as shown in SEQ ID NO. 71.
18. The composition of claim 2, wherein the targeting moiety is
coupled to the anti-microbial peptide moiety via a peptide
linker.
19. The composition of claim 1, wherein the anti-microbial peptide
moiety comprises a peptide selected from the group consisting of
alexomycin, andropin, apidaecin, bacteriocin, .beta.-pleated sheet
bacteriocin, bactenecin, buforin, cathelicidin, .alpha.-helical
clavanin, cecropin, dodecapeptide, defensin, .beta.-defensin,
.alpha.-defensin, gaegurin, histatin, indolicidin, magainin, nisin,
protegrin, ranalexin, and tachyplesin.
20. The composition of claim 1, wherein the anti-microbial peptide
moiety comprises a peptide selected from the group consisting of
histatin 5, dhvar1, protegrin PG-1, and novispirin G10.
21. The composition of claim 1, wherein the target microbial
organism is selected from the group consisting of bacteria,
ricketsia, fungi, yeasts, protozoa, and parasites.
22. The composition of claim 1, wherein the target microbial
organism is a cariogenic organism.
23. The composition of claim 1, wherein the target microbial
organism is Streptococcus mutans.
24. The composition of claim 1, wherein the target microbial
organism is selected from the group consisting of Escherichia coli,
Shigella dysenteriae, Salmonella typhimurium, Streptococcus
pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa.
25. The composition of claim 24, wherein the anti-microbial peptide
moiety comprises a peptide selected from the group consisting of
buforin, cecropin, indolicidin, and nisin.
26. The composition of claim 1, wherein the target microbial
organism is selected from the group consisting of Escherichia coli,
Shigella dysenteriae, Salmonella typhimurium, Streptococcus
pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Candida
albicans, Cryptococcus neoformans, Candida krusei, and Helicobacter
pylori.
27. The composition of claim 26, wherein the anti-microbial peptide
moiety comprises a peptide selected from the group consisting of
magainin and renalexin.
28. A method of treating a target microbial organism infection
comprising administering to a subject in need of such treatment an
effective amount of the composition of claim 1.
29. The method of claim 28, wherein the target microbial organism
infection is on a mucosal surface.
30. The method of claim 28, wherein the target microbial organism
infection is on a surface containing biofilm.
31. The method of claim 29, wherein the mucosal surface is selected
from the group consisting of mouth, vagina, gastrointestinal tract,
and esophageal tract.
32. The method of claim 28, wherein the target microbial organism
infection is a S. mutans infection in a mouth.
33. The method of claim 28, wherein the target microbial organism
infection is a Caudida albicans infection in vagina.
34. The method of claim 28, wherein the target microbial organism
infection is an infection in gastrointestinal tract selected from
the group consisting of a Helicobacter pylori infection,
Campylobacter jerjuni infection, Vibrio cholerae infection,
salmonella infection, Shigella infection, and Escherichia coli
infection.
35. The method of claim 28, wherein the target microbial organism
infection is an oral infection selected from the group consisting
of porphyromonas gingivalis, Actinomyces, Veillonella spirochetes,
and gram-negative flora infection.
36. The method of claim 28, wherein the target microbial organism
infection is an Clostridium difficile infection in gastrointestinal
tract or esophageal tract.
37. A targeting peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO. 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, and 61.
38. A targeting peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO. 24, 25, 26, 27, 28, 29, 30,
31, 32, and 33, wherein the targeting peptide specifically binds to
a microorganism of Pseudomonas.
39. The targeting peptide of claim 38, wherein the targeting
peptide specifically binds to P. aeruginosa.
40. A targeting peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO. 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, wherein the
targeting peptide specifically binds to a microorganism of
Staphylococcus.
41. The targeting peptide of claim 40, wherein the targeting
peptide specifically binds to S. aureus.
42. A targeting peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO. 52, 53, 54, 55, 56, 57, 58,
59, 60, and 61, wherein the targeting peptide specifically binds to
a microorganism of E. coli.
43. The targeting peptide of claim 42, wherein the targeting
peptide specifically binds to E. coli DH5.alpha..
44. A targeting peptide comprising an amino acid sequence as shown
in SEQ ID NO. 61.
45. The targeting peptide of claim 37 which is operably linked to a
detectable moiety.
46. The targeting peptide of claim 37 which is operably linked to
an anti-microbial agent.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/077,624, filed on Feb. 14, 2002 which is a
continuation-in-part of U.S. application Ser. No. 09/910,358, filed
on Jul. 19, 2001 which is a continuation-in-part of U.S.
application Ser. No. 09/378,577, filed on Aug. 20, 1999 all of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of
anti-microbial treatment and more specifically to targeted
anti-microbial treatment by chimeric constructs.
BACKGROUND OF THE INVENTION
[0003] The Centers for Disease Control estimates that half of more
than 100 million annual prescriptions of antibiotics are
unnecessary. As a result, microbes have, in many cases, adapted and
are resistant to antibiotics due to constant exposure and improper
use of the drugs. It is estimated that the annual cost of treating
drug resistant infections in the United States is approximately $5
billion. This continued emergence of anti-microbial-resistant
bacteria, fungi, yeast and parasites has encouraged efforts to
develop other agents capable of killing pathogenic microbes.
Furthermore, there are urgent needs for target-specific
anti-microbial agents since many microbial pathogens reside with
non-harmful commensal bacteria that are important for the health of
the human host.
[0004] Recent scientific studies have revealed a class of naturally
occurring anti-microbial peptides in humans, other mammals, plants,
insects and other organisms. A negative aspect of treatment with
antibiotics or anti-microbial peptides is their indiscriminate
killing or inhibition of a broad spectrum of microorganisms. The
human body is home to millions of different bacteria, many of which
are vital for optimal health. Overuse of broad-spectrum antibiotics
can seriously disrupt the ecology of the normal human microbiota
rendering humans more susceptible to bacterial, yeast, viral, and
parasitic infections. This effect is also seen with administration
of anti-microbial peptides. For example, the antibiotic peptide
histatin kills most gram-positive bacteria in the oral cavity. Thus
general administration of histatin can lead to undesirable effects
by allowing the overgrowth of gram-negative bacteria, such as
Actinobacillus sp or Fusobacterium sp, many of which may cause
periodontal diseases. Accordingly, histatin is not useful by itself
for prevention of dental caries.
[0005] Another disadvantage of administration of anti-microbial
peptides is their ability to damage host cells at higher
concentrations since these positively charged peptides can also
penetrate and disrupt eukaryotic cell membranes.
[0006] Previous efforts to deliver of pharmaceutically active
agents to specific targets relied principally on chemically
conjugating a pharmaceutically active agent to a targeting
component. For example Shih et al. U.S. Pat. No. 5,057,313 refers
to targeting delivery of drugs, toxins and chelators to specific
sites in an organism by loading a therapeutic or diagnostic
component onto a polymeric carrier, followed by conjugation of the
carrier to a targeting antibody. Hansen, U.S. Pat. No. 5,851,527
claims a similar invention.
[0007] A drawback to this approach is that the non-specific linkage
of the pharmaceutical reagents to unknown sites on the antibody
molecule used for targeting may interfere with delivery of the
therapeutic agents. See Rodwell et al., U.S. Pat. No. 4,671,958.
Moreover, chemical modification of a targeting antibody by the
nonspecific reactions during conjugation may substantively alter
the antibody itself, thereby affecting its binding to targets.
Furthermore, chemical linkage is very inefficient and the result is
non-uniform, making the technique very difficult to use in
practice.
[0008] More recently, there have been a number of reports of the
use of recombinant techniques to produce fusion proteins for the
treatment of disease. See Penichet and Morrison, J. Immunological
Methods, 248:91-101 (2001) for review. Penichet et al. discuss
efforts to treat malignant disease using a genetically engineered
protein construct including an immunological component that binds
specifically to tumor cells and a cytokine capable of eliciting
significant antitumor activity. See, e.g. Pastan et al. U.S. Pat.
No. 5,981,726, and Fell, Jr. et al., U.S. Pat. No. 5,645,835.
[0009] However, to date there have not been any reports of
directing anti-microbial agents to infected regions of humans or
animals using target-specific molecules. There is a need in the art
to provide methods and compositions useful for treatment of
microbial organisms and microbially mediated diseases, especially
microbial diseases of mucosal surfaces that are not readily
accessible by normal anti-microbial mechanisms provided by the
immune system.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery that
anti-microbial peptides can be purposely directed to specific
microbial organisms by a targeting moiety connected to the
anti-microbial peptides. Accordingly the present invention provides
a composition that has an anti-microbial effect on a targeted
microbial organism. The present invention also provides methods of
treating a microbial infection, e.g., on mucosal surfaces by using
the compositions provided by the present invention.
[0011] In one embodiment, the present invention provides a
composition useful for treatment of microbial organisms. The
composition comprises a targeting moiety and an anti-microbial
peptide moiety, wherein the targeting moiety is coupled to the
anti-microbial peptide moiety and recognizes a target microbial
organism and wherein the composition has an anti-microbial effect
on the target microbial organism.
[0012] In another embodiment, the composition comprises a targeting
moiety and an anti-microbial peptide moiety, wherein the targeting
moiety is a peptide, e.g., polypeptide or small peptide and is
fused in-frame with the anti-microbial peptide moiety. Such
composition can be produced recombinantly using an expression
system, e.g., bacterial, yeast, or eukaryotic cell expression
system, without having to deal with problems associated with
chemical or physical linkages.
[0013] In another embodiment, the present invention provides a
method of treating a target microbial organism infection. The
method comprises administering to a subject in need of such
treatment an effective amount of the composition of the present
invention.
[0014] In yet another embodiment, the present invention provides a
targeting peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48,
49, 50, 51, 52, 53, 45, 55, 56, 57, 58,59, 60, and 61.
[0015] In still another embodiment, the present invention provides
a targeting peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31,
32, and 33, wherein the targeting peptide specifically binds to a
microorganism of Pseudomonas.
[0016] In yet another embodiment, the present invention provides a
targeting peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO. 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, wherein the targeting
peptide specifically binds to a microorganism of
Staphylococcus.
[0017] In another embodiment, the present invention provides a
targeting peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO. 52, 53, 45, 55, 56, 57, 58, 59,
60, and 61, wherein the targeting peptide specifically binds to a
microorganism of E. coli.
SUMMARY OF THE FIGURES
[0018] FIG. 1 shows a schematic diagram of the sequential PCR
reactions used to assemble the heavy chain portion of the
antibody-based fusion protein.
[0019] FIG. 2 shows the sequences (SEQ ID NOS: 7-13) of the primers
used in the sequential PCR reactions in embodiments of the present
invention.
[0020] FIG. 3 shows the nucleotide sequence (SEQ ID NO: 1) encoding
the anti-microbial peptide, histatin 5, the linker peptide, and the
variable region of the heavy chain derived from the SWLA3
monoclonal antibody together with the amino acid sequence (SEQ ID
NO: 2).
[0021] FIG. 4 shows the nucleotide sequence (SEQ ID NO: 3) encoding
the anti-microbial peptide, dhvar 1, the linker peptide, and the
variable region of the heavy chain derived from the SWLA3
monoclonal antibody together with the amino acid sequence (SEQ ID
NO: 4).
[0022] FIG. 5 shows the schematic diagram of making a
minibody-anti-microbial peptide fusion protein.
[0023] FIG. 6 shows killing kinetics of G10 against Pseudomonas
mendocina.
[0024] FIG. 7 shows killing kinetics of G10CatC against Pseudomonas
mendocina
[0025] FIG. 8 shows killing kinetics of G10CatN against Pseudomonas
mendocina.
[0026] FIG. 9 shows killing kinetics of G10 against Pseudomonas
aeruginosa PAK.
[0027] FIG. 10 shows killing kinetics of G10CatC against
Pseudomonas aeruginosa PAK.
[0028] FIG. 11 shows long-term killing kinetics of G10 and G10CatC
against Pseudomonas mendocina.
[0029] FIG. 12 shows selective killing of Pseudomonas mendocina by
G10CatC in mixed culture.
[0030] FIG. 13 shows nonselective killing of Pseudomonas mendocina
by G10 in mixed culture.
[0031] FIG. 14 shows selective killing of PAK by G10CatC in mixed
culture.
[0032] FIG. 15 shows nonselective killing of PAK by G10 in mixed
culture.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] The present invention relates in general to the targeted
anti-microbial effects using a composition, e.g., a chimeric
construct containing a targeting moiety and an anti-microbial
peptide moiety. The present invention also provides methods of
treating a microbial infection using the compositions provided by
the present invention.
[0034] According to the present invention, a targeting moiety can
be any suitable structure that recognizes and binds to a target
microbial organism. For example, a targeting moiety can be a
polypeptide, peptide, small molecule, ligand, receptor, antibody,
protein, or portions thereof that specifically interacts with a
target microbial organism, e.g., the cell surface appendages such
as flagella and pili, and surface exposed proteins, lipids and
polysaccharides of a target microbial organism.
[0035] In one embodiment, the targeting moiety of the present
invention is a monoclonal antibody or one of various forms of a
monoclonal antibody that specifically recognizes an epitope or
antigen of a target microbial organism. Such epitope or antigen
usually is species-specific and located on the surface of a target
microbial organism. A monoclonal antibody or various forms thereof
in a targeting moiety can direct an anti-microbial peptide moiety
to its target site. Furthermore, it may also provide anti-microbial
effect in addition to the effect provided by the anti-microbial
peptide moiety since such monoclonal antibody may engage an immune
system and elicit an antibody-associated immune response, e.g.,
humoral immune response.
[0036] A monoclonal antibody specific to a microbial organism can
be made using any methods readily available to one skilled in the
art. For example, as described in the U.S. Pat. No. 6,231,857
(incorporated herein by reference) three monoclonal antibodies,
i.e., SWLA1, SWLA2, and SWLA3 have been made against S. mutans.
Monoclonal antibodies obtained from non-human animals to be used in
a targeting moiety can also be humanized by any means available in
the art to decrease their immunogenicity and possibly increase
their ability to elicit anti-microbial immune response of a
human.
[0037] Various forms of a monoclonal antibody include, without
limitation, scFv, minibody, Di-miniantibody, Tetra-miniantibody,
(scFv).sub.2, Diabody, scDiabody, Triabody, Tetrabody, and Tandem
diabody. A scFv usually comprises a single chain containing the
variable regions of a light chain and a heavy chain, optionally
joined via a linker. A minibody usually comprises the variable
regions of a light chain and a heavy chain, e.g., scFv joined to a
heavy chain constant region, e.g., about 20 amino acids or the
third constant domain, C.sub.H3 domain, either directly or via a
linker, e.g., about 10 to 25 amino acids. A minibody can be readily
made by expressing its encoding sequence in any suitable host such
as E. coli, yeast, or eukaryotic cell lines. A readily prepared
version of a minibody usually forms a disulfide-linked dimer by
virtue of the constant region, e.g., C.sub.H3 domain and a
cysteine-containing linker. Various forms of a monoclonal
antibodies are described in Little et al., Immunology Today,
21:364-370 (2000), which is incorporated herein by reference.
[0038] Alternatively, the targeting moiety of the present invention
can include all or a portion of one or more variable regions that
are capable of specifically recognizing or binding to a target
microbial organism and optionally a portion of constant regions
that is sufficient for dimerization. For example, the variable
region of a heavy chain has three complementarity determining
regions (CDRs) and is capable of binding to an antigen. One skilled
in the art can readily assess the minimum variable regions required
of any particular monoclonal antibody for antigen or epitope
binding.
[0039] According to another embodiment of the present invention, a
targeting moiety can be a targeting peptide capable of specifically
binding to a microorganism, e.g., a target microbial organism. In
one embodiment, the targeting peptide provided by the present
invention can be identified via screening peptide libraries. For
example, a phage display peptide library can be screened against a
target microbial organism or a desired antigen or epitope thereof.
Any peptide identified through such screening can be used as a
targeting peptide for the target microbial organism.
[0040] In another embodiment, the targeting peptide provided by the
present invention is a peptide capable of specifically binding to
Pseudomonas, especially P.aeruginosa. Such targeting peptide
includes, without any limitation, a peptide containing an amino
acid sequence as shown in SEQ ID NO. 24, 25, 26, 27, 28, 29, 30,
31, 32, or 33.
[0041] In yet another embodiment, the targeting peptide provided by
the present invention is a peptide capable of specifically binding
to Staphylococcus, especially S. aureus. Such targeting peptide
includes, without any limitation, a peptide containing an amino
acid sequence as shown in SEQ ID NO. 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51.
[0042] In still another embodiment, the targeting peptide provided
by the present invention is a peptide capable of specifically
binding to E. coli. Such targeting peptide includes, without any
limitation, a peptide containing an amino acid sequence as shown in
SEQ ID NO. 52, 53, 54, 55, 56, 57, 58, 59, or 60.
[0043] According to the present invention, the targeting peptide of
the present invention can also be a peptide obtained based on
rational design. For example, one can design a targeting peptide
based on the biochemical and biophysical characteristics of amino
acids and the surfaces of microorganisms. In general, positively
charged peptides are likely to bind negatively charged components
on the cell surface and vice versa. Similarly, hydrophobic peptides
may bind to hydrophobic pockets on the cell surface based on
hydrophobic interactions while secondary or tertiary structure of a
peptide may fit into certain structures on the surface of a
microorganism.
[0044] In one embodiment, the targeting peptide provided by the
present invention is a peptide containing an amino acid sequence as
shown in SEQ ID NO. 61, 62, 63, 64, 65, 66, 67, or 68. In another
embodiment, the targeting peptide provided by the present invention
is a peptide containing an amino acid sequence as shown in SEQ ID
NO. 61 and capable of specifically binding to Pseudomonas, or E.
coli.
[0045] The targeting peptides provided by the present invention can
be naturally or non-naturally occurring peptides. For example, the
targeting peptides provided by the present invention can be
recombinantly made, chemically synthesized, or naturally existing.
In one embodiment, the targeting peptide contains an amino acid
sequence that constitutes an internal part of a naturally occurring
polypeptide. In another embodiment, the targeting peptide contains
an amino acid sequence encoded by a sequence naturally existing in
a genome and such amino acid sequence is not adjacent to any amino
acid sequence naturally adjacent to it, e.g., such amino acid
sequence is adjacent to a heterologous sequence in the targeting
peptide.
[0046] The targeting peptide provided by the present invention can
also include a peptide having an amino acid sequence that is
derived or modified from a targeting amino acid sequence
specifically illustrated in the present invention, provided that
the derived or modified sequence still maintains or has an enhanced
specificity with respect to its target microbial organism. For
example, the targeting amino acid sequence can be structurally
modified via deletion, mutation, addition of amino acids or other
structural entities, or any other structural changes as long as
these changes do not alter or adversely affect the binding ability
of the targeting amino acid sequence to its target microbial
organism.
[0047] In one embodiment, the modifications to the original
targeting amino acid sequence do not alter its core sequence, e.g.
the consensus sequence among a group of targeting amino acid
sequences provided by the present invention. For example, the
consensus sequence among targeting amino acid sequences of SEQ ID
NO. 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33 is
V/Q/H-P/H-H-E-F/Y-K/H-H/A-L/H-X-X-K/R-P/L (SEQ ID NO. 14), and
according to the present invention any modification to a targeting
amino acid sequence of SEQ ID NO. 24, 25, 26, 27, 28, 29, 30, 31,
32, and 33 should not alter the consensus sequence contained
therein, e.g., should not alter the
V/Q/H-P/H-H-E-F/Y-K/H-H/A-L/H-X-X-K/R-P/L (SEQ ID NO. 14) sequence
contained therein.
[0048] The targeting peptide provided by the present invention is
useful for delivery of various entities to a desired locus. For
example, the targeting peptide provided by the present invention
can be used to deliver anti-microbial agents such as anti-microbial
peptides and detectable agents such as imaging agents, which are
detectable either directly or via a secondary imaging agent. For
example, the detectable agent to be delivered can be an agent which
can be used with an imaging technique such as magnetic resonance
imaging (MRI), positron emission tomography (PET),
computer-assisted tomography (CAT), X-ray, fluoroscopy and single
photon emission computerized tomography.
[0049] The targeting moiety of the present invention can also be a
ligand, receptor, or fragment thereof that specifically recognizes
a target microbial organism. For example, the targeting moiety of
the present invention can be glucan binding proteins of
Streptococcus mutans that can specifically bind insoluble glucans
on the surface of S. mutans.
[0050] The composition of the present invention can contain one or
more targeting moieties capable of targeting the same or different
target microbial organisms. In one embodiment, the composition of
the present invention contains one or more targeting moieties
capable of targeting different sites or structures of the same
target microbial organism. Such composition is useful for
preventing resistance of a target microbial organism to the
composition.
[0051] According to the present invention, an anti-microbial
peptide moiety of the composition of the present invention
comprises one or more anti-microbial peptides. In general, any
known or later discovered anti-microbial peptides can be used for
the compositions of the present invention. Anti-microbial peptides
are various classes of peptides, e.g., peptides originally isolated
from plants as well as animals. In animals, anti-microbial peptides
are usually expressed by various cells including neutrophils and
epithelial cells. In mammals including human, anti-microbial
peptides are usually found on the surface of the tongue, trachea,
and upper intestine.
[0052] Naturally occurring anti-microbial peptides are generally
amphipathic molecules that contain fewer than 100 amino acids. Many
of these peptides generally have a net positive charge (i.e.,
cationic) and most form helical structures. It is generally
believed that these peptides' anti-microbial efficacy is in their
ability to penetrate and disrupt the microbial membranes, thereby
killing the microbe or inhibiting its growth.
[0053] The anti-microbial activities of the anti-microbial peptides
of the present invention include, without limitation,
antibacterial, antiviral, or antifungal activities. For example,
one well-known class of anti-microbial peptides are the
tachyplesins which are described as having antifungal and
antibacterial activities. Andropin, apidaecin, bactencin, clavanin,
dodecappeptide, defensin, and indolicidin are anti-microbial
peptides having antibacterial activities. Buforin, nisin and
cecropin peptides have been demonstrated to have anti-microbial
effects on Escherichia. coli, Shigella disenteriae, Salmonella
typhimurium, Streptococcus pneumoniae, Staphylococcus aureus, and
Pseudomonas aeroginosa. Magainin and ranalexin peptides have been
demonstrated to have anti-microbial effects on the same organsims,
and in addition have such effects on Candida albicans, Cryptococcus
neoformans, Candida krusei, and Helicobacter pylori. Magainin has
also been demonstrated to have anti-microbial effects on herpes
simplex virus. Alexomycin peptides have been demonstrated to have
anti-microbial effects on Campylobacter jejuni, Moraxella
catarrhalis and Haemophilus inflluenzae while a defensin and .beta.
pleated sheet defensin peptides have been shown to have
anti-microbial effects on Streptococcus pneumoneae.
[0054] Histatin peptides and the derivatives thereof are another
class of anti-microbial peptides, which have antifungal and
antibacterial activities against a variety of organisms including
Streptococcus mutans. MacKay, B. J. et al., Infect. Immun.
44:695-701 (1984); Xu, et al., J. Dent. Res. 69:239 (1990).
[0055] In one embodiment, the anti-microbial peptide moiety of the
present invention contains one or more anti-microbial peptides from
a class of histatin peptides and the derivatives thereof. For
example, the anti-microbial peptide moiety of the present invention
contains one or more derivatives of histatin including, without
limitation, histatin 5 having an amino acid sequence as shown in
SEQ ID NO. 5 or dhvar 1 having an amino acid sequence as shown in
SEQ ID NO. 6.
[0056] In another embodiment, the anti-microbial peptide moiety of
the present invention contains one or more anti-microbial peptides
from a class of protegrins and the derivatives thereof. For
example, the anti-microbial peptide moiety of the present invention
contains protegrin PG-1 having an amino acid sequence
RGGRLCYCRRRFCVCVGR as shown in SEQ ID NO. 15. The protegrin
peptides have been shown to have anti-microbal effects on
Streptococcus mutans, Neisseria gonorrhoeae, Chlamydia trachomatis
and Haempohilus influenzae. Protegrin peptides are described in the
U.S. Pat. Nos. 5,693,486, 5,708,145, 5,804,558, 5,994,306, and
6,159,936, all of which are incorporated herein by reference.
[0057] In yet another embodiment, the anti-microbial peptide moiety
of the present invention contains one or more anti-microbial
peptides from a class of novispirin and the derivatives thereof as
described in Sawai et al., "Impact of Single-Residue Mutations on
the Structure and Function of Ovispirin/Novispirin Antimicrobial
Peptides." Protein Engineering (in press). For example, the
anti-microbial peptide moiety of the present invention contains
novispirin G10 having an amino acid sequence KNLRRIIRKGIHIIKKYG as
shown in SEQ ID NO. 16 for treating cariogenic organisms, e.g.,
Streptococcus mutans.
[0058] In still another embodiment, the anti-microbial peptide
moiety contains one or more anti-microbial peptides including,
without limitation, alexomycin, andropin, apidaecin, bacteriocin,
.beta.-pleated sheet bacteriocin, bactenecin, buforin,
cathelicidin, .alpha.-helical clavanin, cecropin, dodecapeptide,
defensin, .beta.-defensin, .alpha.-defensin, gaegurin, histatin,
indolicidin, magainin, nisin, protegrin, ranalexin, tachyplesin,
and derivatives thereof.
[0059] The anti-microbial peptide moiety of the present invention
can include one or more anti-microbial peptides, which can be the
same or different anti-microbial peptides. The anti-microbial
peptides of the present invention can also be modified, e.g., to
enhance its anti-microbial effectiveness, its cell delivery, its
compatibility with the rest of the composition structure, or the
manipulation of the composition in production.
[0060] The targeting moiety and the anti-microbial peptide moiety
of the present invention can be coupled by various means known to
one skilled in the art. For example, the targeting moiety and the
anti-microbial peptide moiety can be covalently coupled or
connected by a peptide linker; and the composition so formed can be
constructed through molecular cloning and overexpressed and
purified as one polypeptide unit in a bacterial, yeast, or
eukaryotic cell expression system. Any peptide linker can be used
to connect the targeting moiety and the anti-microbial peptide
moiety of the present invention. In one embodiment, the peptide
linker does not interfere or inhibit the activity of the targeting
moiety or the anti-microbial peptide moiety. In another embodiment,
the peptide linker is from about 10 to 60 amino acids, from about
15 to 25 amino acids, or about 15 amino acids.
[0061] An anti-microbial peptide can be connected to a targeting
moiety at either or both ends of the targeting moiety. In one
embodiment, a targeting moiety is a peptide or polypeptide which
can be fused in frame at N-terminal, C-terminal, or both ends with
one or more anti-microbial peptides.
[0062] In another embodiment, the targeting moiety is a targeting
peptide containing an amino acid sequence as shown in SEQ ID NO. 61
and is fused in frame at either end of an anti-microbial peptide,
e.g., novispirin G10 as shown in SEQ ID NO. 70 and 71. In yet
another embodiment, the targeting moiety is a targeting peptide
containing an amino acid sequence as shown in SEQ ID NO. 61 and is
fused in frame at the C-terminal end of an anti-microbial peptide,
e.g., novispirin G10 as shown in SEQ ID NO. 70.
[0063] The composition of the present invention can be made by any
suitable means known to one skilled in the art. For example, a
nucleotide sequence encoding a targeting moiety ligated to a
nucleotide sequence encoding an anti-microbial peptide moiety,
either directly or via a nucleotide sequence encoding a peptide
linker, can be expressed in an appropriate expression system, e.g.,
a commercially available bacterial, yeast, or eukaryotic cell
expression system. Usually for expressing in a bacterial expression
system, an autocatalytic protein, e.g., intein and a chitin-binding
domain (CBD) are used for purification purpose. For expressing in a
yeast expression system, a pheromone factor a is usually fused to
the N-terminal of a coding sequence while a myocin-his tag is fused
to the C-terminal of the coding sequence for easy handling of the
expressed product during the purification process.
[0064] In one embodiment of the present invention, a commercially
available yeast expression system is modified, e.g., proteins used
for bacterial expression systems are used for yeast expression. For
example, a sequence encoding the composition of the present
invention is fused with a sequence encoding pheromone factor
.alpha. and a sequence encoding intein and CBD and is expressed in
a yeast expression system.
[0065] The compositions of the present invention can be used to
treat any target microbial organisms. For example, the target
microbial organism of the present invention can be any bacteria,
rickettsia, fungi, yeasts, protozoa, or parasites. In one
embodiment, the target microbial organism is a cariogenic organism,
e.g., Streptococcus mutans.
[0066] In another embodiment, the target microbial organisms of the
present invention include, without limitation, Escherichia. coli,
Candida, Salmonella, Staphylococcus, and Pseudomonas, especially
Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia
trachomatis, Clostridium difficile, Cryptococcus neoformans,
Haempohilus influenzae, Helicobacter pylor, Moraxella catarrhalis,
Neisseria gonorrhoeae, Pseudomonas aeroginosa, Salmonella
typhimurium, Shigella disenteriae, Staphylococcus aureus, and
Streptococcus pneumoniae.
[0067] According to another feature of the present invention, the
compositions of the present invention provide anti-microbial effect
to target microbial organisms and can be used to treat a target
microbial organism infection. An anti-microbial effect includes
inhibiting the growth or killing of the target microbial organisms,
or interfering with any biological functions of the target
microbial organisms.
[0068] In general, the compositions of the present invention can be
used to treat a target microbial organism infection at any place in
a host, e.g., at any tissue including surfaces of any implant. In
one embodiment, the compositions of the present invention are used
to treat a target microbial organism infection on a mucosal surface
or a surface containing a biofilm. A mucosal surface usually
harbors a broad spectrum of microbial organisms, e.g., existing as
a biofilm and prefers a treatment that is least disturbing to the
balance of the entire microbial organism population, e.g., specific
to pathogenic microbial organisms and has minimum effect on the
non-pathogenic microbial population.
[0069] For example, in human mouth there usually exist many
different microbes including yeasts and bacteria. Most of the
bacteria are non-harmful commensal bacteria that are essential for
maintaining a healthy and normal microbial flora to prevent the
invasion and establishment of other pathogenic microbial organisms,
e.g., yeast infection. Administering the composition of the present
invention targets specifically to cariogenic organisms, e.g.
Streptococcus mutans and will have minimum effect on non-targeted
microbial organisms, thus will not have an undesirable effect by
non-targeted microbial organisms.
[0070] Many places in an animal or human body have mucosal surfaces
colonized by multiple species microbial biofilms and can be treated
with the compositions of the present invention to provide targeted
anti-microbial effect. For example, mouth, vagina, gastrointestinal
(GI) tract, esophageal tract, respiratory tract, implants, all of
which can have microbial organism infection on its mucosal
surfaces.
[0071] In particular, S. mutans infection is commonly found in
mouth and causes. dental caries. Porphyromonas gingivalis, various
Actinomyces species, Veillonella, spirochetes, and black-pigmented
bacteroides are commonly associated with infections of gingival and
surrounding connective tissues, which cause periodontal diseases.
Streptococcus pneumoniae, nontypeable Haemophilius influenza, or
Moraxella cararrhalis infection is commonly found in acute otitis
media (AOM) and otitis media effusion (OME) as complications of
upper respiratory infections in young children.
[0072] Helicobacter pylori (H. pylori) bacteria are found in the
gastric mucous layer or adherent to the epithelial lining of the
stomach, and cause more than 90% of duodenal ulcers and up to 80%
of gastric ulcers. Other GI tract infections include, without
limitation, campylobacter bacterial infection, primarily
Campylobacter jejuni associated with diarrhea, cholera caused by
Vibrio cholerae serogroups, salmonellosis caused by bacteria
salmonella such as S. Typhimurium and S. enteritidis, shigellosis
caused by bacteria Shigella, e.g., Shigella dysenteriae and
traveler's diarrhea caused by enterotoxigenic Escherichia coli
(ETEC). Clostridium difficile infection is also commonly found in
gastrointestinal tract or esophageal tract.
[0073] Pseudomonas organisms have been associated with
common-source nosocomial outbreaks; in addition, they have been
incriminated in bacteremia, endocarditis, and osteomyelitis in
narcotic addicts. Infections with Pseudomonas organisms can also
occur in the ear, lung, skin, or urinary tract of patients, often
after the primary pathogen has been eradicated by antibiotics.
Serious infections are almost invariably associated with damage to
local tissue or with diminished host resistance. Patients
compromised by cystic fibrosis and those with neutropenia appear at
particular risk to severe infection with P. aeruginosa. Premature
infants; children with congenital anomalies and patients with
leukemia; patients with burns; and geriatric patients with
debilitating diseases are likely to develop Pseudomonas infections.
The organism is prevalent in urine receptacles and on catheters,
and on the hands of hospital staff.
[0074] The staphylococci, of which Staphylococcus aureus is the
most important human pathogen, are hardy, gram-positive bacteria
that colonize the skin of most human beings. If the skin or mucous
membranes are disrupted by surgery or trauma, staphylococci may
gain access to and proliferate in the underlying tissues, giving
rise to a typically localized, superficial abscess. Although these
cutaneous infections are most commonly harmless, the multiplying
organisms may invade the lymphatics and the blood, leading to the
potentially serious complications of staphylococcal bacteremia.
[0075] These complications include septic shock and serious
metastatic infections, including endocarditis, arthritis,
osteomyelitis, pneumonia, and abscesses in virtually any organ.
Certain strains of S. aureus produce toxins that cause skin rashes
or that mediate multisystem dysfunction, as in toxic shock
syndrome. Coagulase-negative staphylococci, particularly S.
epidermidis, are important nosocomial pathogens, with a particular
predilection for infecting vascular catheters and prosthetic
devices. S. saprophyticus is a common cause of urinary tract
infection.
[0076] Yeast or Candida infections (Candidiasis) typically occur
either orally (Oropharyngeal Candida or OPC) or vaginally
(Vulvovaginal Candida or VVC). Candidiasis is caused by a shift in
the local environment that allows Candida strains (most commonly
Candida albicans) already present on skin and on mucosal surfaces
such as mouth and vagina to multiply unchecked. Gonorrhea,
chlamydia, syphilis, and trichomoniasis are infections in the
reproductive tract, which cause sexually transmitted diseases,
e.g., pelvic inflammatory disease.
[0077] The compositions of the present invention can be
administered to various mucosal or biofilm surfaces, e.g., the
mucosal surfaces described above, with each composition containing
a targeting moiety corresponding to one or more specific microbial
organisms of the infection, e.g., the microbial organisms described
above.
[0078] The composition of the present invention can also be
administered to various biofilm surfaces outside of a human body,
e.g., industrial applications. For example, in food processing
industry the composition of the present invention can be
administered to food processing equipments or food itself to
prevent infections related to food consumption, e.g., Salmonella in
a poultry processing facility.
[0079] The compositions of the present invention useful for
treating target microbial organism infection can be administered
alone, in a composition with a suitable pharmaceutical carrier, or
in combination with other therapeutic agents. An effective amount
of the compositions to be administered can be determined on a
case-by-case basis. Usually the dosage required is lower than the
dosage required for an anti-microbial peptide administered without
being linked to a targeting moiety, e.g., 10.sup.-1 lower. Factors
to be considered usually include age, body weight, stage of the
condition, other disease conditions, duration of the treatment, and
the response to the initial treatment.
[0080] Typically, the compositions are prepared as a topical or an
injectable, either as a liquid solution or suspension. However,
solid forms suitable for solution in, or suspension in, liquid
vehicles prior to injection can also be prepared. The composition
can also be formulated into an enteric-coated tablet, gel capsule
or microsphere formulation according to known methods in the
art.
[0081] The compositions of the present invention may be
administered in any way which is medically acceptable which may
depend on the disease condition or injury being treated. Possible
administration routes include injections, by parenteral routes such
as intravascular, intravenous, intraepidural or others, as well as
oral, nasal, ophthalmic, rectal, topical, or pulmonary, e.g., by
inhalation. The compositions may also be directly applied to tissue
surfaces. Sustained release, pH dependent release, or other
specific chemical or environmental condition mediated release
administration is also specifically included in the invention, by
such means as depot injections or erodible implants.
[0082] In one embodiment, the compositions of the present invention
are used to treat or prevent cariogenic organism infections, e.g.,
S. mutans infection associated with dental caries and are prepared
as additives to food or any products having direct contact to an
oral environment, especially an oral environment susceptible to
dental caries. For example, to treat or prevent dental caries one
or more compositions of the present invention can be formulated
into a baby formula, mouthwash, lozenges, gel, varnish, toothpaste,
toothpicks, tooth brushes, or other tooth cleansing devices,
localized delivery devices such as sustained release polymers or
microcapsules, oral irrigation solutions of any kind whether
mechanically delivered or as oral rinses, pacifiers, and any food
including, without limitation, chewing gums, candies, drinks,
breads, cookies, and milk.
EXAMPLES
[0083] The following examples are intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
Example 1
Construction and Expression of a Histatin 5 and Dhvar 1/SWLA3
Chimeric Antibody Fusion Protein with Activity Against S.
mutans
[0084] a. Construction of an Expression Vector for an
Antibody-Based Fusion Protein
[0085] The construct that is ultimately cloned into an IgG.sub.1
expression vector and leads to the expression of the targeted
anti-microbial fusion protein was assembled according to the
following method (see FIG. 1). The construct was assembled using
sequential PCR and restriction enzymes techniques. The recognition
sequence of the fusion protein was derived from heavy chain
sequences of SWLA3, produced by hybridoma ATCC HB 12558. See Shi,
U.S. Pat. No. 6,231,857, the disclosure of which is incorporated
herein by reference, and U.S. patent application Ser. Nos.
09/378,577 and 09/881,823. Sequences encoding histatin 5 or dhvar1
were inserted upstream of the variable region of the heavy chain of
SWLA3. The amino acid sequences used for histatin 5 and dhvar 1 are
listed below:
1 Histatin (SEQ ID NO: 5) DSHAKRHHGY KRKFHEKHHS HRGY 5 Dhvar 1 (SEQ
ID NO: 6) KRLFKELKFS LRKY.
[0086] The source signal peptide was added upstream of the histatin
5 or dhvar1, and a glycine/serine linker was added to separate the
fusion protein from the variable region of the heavy chain
(V.sub.H) of the antibody. See FIG. 3 for the nucleic acid and
encoded amino acid sequence for the histatin 5/SWLA3 V.sub.H and
FIG. 4 for the respective dhvar 1/SWLA3 V.sub.H sequences.
Sequential PCR reactions were used to complete the construct
according to the following method (see FIG. 2 for the nucleic acid
sequence of the primers used):
[0087] 1. In the first PCR reaction a plasmid carrying the V.sub.H
of SWLA3 was used as the template with primer sets 986+452
(histatin 5) or 989+452 (dhvar1). This reaction replaced the signal
peptide in the original gene with the linker peptide at the 5' end
of the VH and inserted a restriction site at the 3' end. The
products of this reaction were isolated and used as a template in
the second PCR reaction.
[0088] 2. Using primer sets 987+452 (histatin 5) or 990+452
(dhvar1) in the second PCR reaction added the anti-microbial
peptide upstream from the linker peptide. The restriction site at
the 3' end was maintained. The products from this reaction were
isolated and used as the template in the third PCR reaction.
[0089] 3. With primer sets 988+452 (histatin 5) or 991+452 (dhvar1)
a signal peptide and restriction site were added upstream from the
anti-microbial peptide. The restriction site at the 3' end was
maintained. Products from the third PCR were isolated.
[0090] 4. Isolated products from the third PCR reaction were then
cloned into Invitrogen's PCR2.1 vector via TOPO Cloning Kit and
sequenced.
[0091] 5. After the sequences of the two clones were confirmed, the
inserts were moved into the IgG.sub.1 PCR expression vector (pAH
4604) as an NheI/EcoRV fragment.
[0092] 6. The final expression vectors for the histatin 5 and dhvar
1 antibody fusion proteins were named pAH 5993 and pAH 5994
respectively.
[0093] PCR conditions used were:
[0094] 1. Denature @ 94.degree. C. for 40 sec.
[0095] 2. Anneal @ 60.degree. C. for 40 sec.
[0096] 3. Extend @ 72.degree. C. for 40 sec.
[0097] 4. Amplify for 30 cycles
[0098] 5. Final Extension at 72.degree. C. for 10 min.
[0099] FIG. 3 shows the nucleic acid sequence encoding the histatin
5 fusion to V.sub.H SWLA3 and encoded amino acid sequence (SEQ ID
NOS: 1 and 2) and FIG. 4 which shows the nucleic acid sequence
encoding the dhvar1 fusion to V.sub.H SWLA3 and encoded amino acid
sequence (SEQ ID NOS: 3 and 4). In the figures, the bold sequences
represent the corresponding anti-microbial peptides, the underlined
sequences represent the glycine/serine linker, and the single
bolded underlined base in each sequence represents a silent point
mutation. In the original sequence disclosed in Shi et al. U.S.
patent application Ser. No. 09/881,823, the base is guanine.
[0100] The variable region of the light chain (V.sub.L) from SWLA3
was cloned into a human kappa expression vector named 5940 pAG
according to the method described in Shi et al. U.S. patent
application Ser. No. 09/881,823. Briefly,
[0101] (i) DNA was prepared from the expression vectors and from
the plasmid containing the correct V.sub.L. See Current Protocols
in Immunology, Section 2.12.1 (1994) for detailed information about
the vectors that express the light and heavy chain constant
regions.
[0102] (ii) The expression vector was digested with the appropriate
restriction enzyme. The digests were then electrophoresed on an
agarose gel to isolate the appropriate sized fragment.
[0103] (iii) The plasmid containing the cloned V.sub.L region was
also digested and the appropriate DNA fragment containing the
V.sub.L region was isolated from an agarose gel.
[0104] (iv) The V.sub.L region and expression vector were then
mixed together, T4 DNA ligase was added and the reaction mixture
was incubated at 16.degree. C. over night.
[0105] (v) Competent cells were transfected with the V.sub.L
ligation mixture and the clones expressing the correct ligation
sequence were selected. Restriction mapping was used to confirm the
correct structure.
[0106] b. Transfecting Eukaryotic Cells
[0107] Ten micrograms of DNA from each expression vector, pAH 5993
(histatin 5) or pAH 5994 (dhvar 1) and 5940 pAG, was linearized by
BSPC1 (Stratagene, PvuI isoschizomer) digestion and
1.times.10.sup.7 myeloma cells (SP2/0 or P3.times.63. Ag8.653) were
cotransfected by electroporation. Prior to transfection the cells
were washed with cold PBS, then resuspended in 0.9 ml of the same
cold buffer and placed in a 0.4 cm electrode gap electroporation
cuvette. 960 microF and 200V was used for electroporation. The
shocked cells were then incubated on ice in IMDM medium (Gibco,
N.Y.) with 10% calf serum.
[0108] The transfected cells were plated into 96 well plates at a
concentration of 10000 cells/well. Selective medium including
selective drugs such as histidinol or mycophenolic acid were used
to select the cells which contain expression vectors. After 12
days, the supernatants from growing clones were tested for antibody
production.
[0109] c. Analyses of Histatin-5 and dhvar 1/SWLA3 Chimeric
Antibody Fusion Proteins
[0110] ELISA assay was used to identify transfectomas that secrete
the fusion IgG antibodies. 100 .mu.l of 5 .mu.g/ml goat anti-human
IgG was added to each well of a 96-well ELISA plate and incubated
overnight. The plate was washed several times with PBS and blocked
with 3% BSA. Supernatants from above growing clones were added to
the plate for 2 hours at room temperature to assay for their
reactivity with goat anti-human Ig antibody. Plates were then
washed and anti-human kappa antibody labeled with alkaline
phosphatase diluted 1:10.sup.4 in 1% BSA was added for 1 hour at
37.degree. C. Plates were washed with PBS and para-nitrophenyl
phosphate in diethanolamine buffer (9.6% diethanolamine, 0.24 mM
MgCl.sub.2, pH 9.8) was added. Color development at OD.sub.405 was
indicative of cells producing H.sub.2L.sub.2.
[0111] For the supernatants that produce IgG constant regions,
their reactivity with S. mutans was tested as described in Shi et
al., Hybridoma 17:365-371 (1998). Briefly, bacteria strains listed
in Table 1 were grown in various media suggested by the American
Type Culture Collection. The anaerobic bacteria were grown in an
atmosphere of 80% N.sub.2, 10% CO.sub.2, and 10% H.sub.2 at
37.degree. C. The specificity of antibodies to various oral
bacteria was assayed with ELISA assays. Bacteria were diluted in
PBS to OD.sub.600=0.5, and added to duplicate wells (100 .mu.l) in
96 well PVC ELISA plates preincubated for 4 h with 100 .mu.l of
0.02 mg/ml Poly-L-lysine. These antigen-coated plates were
incubated overnight at 4.degree. C. in a moist box then washed 3
times with PBS and blocked with 0.5% fetal calf serum in PBS and
stored at 4.degree. C. 100 .mu. l of chimeric antibodies at 50
.mu.g/ml were added to the appropriate wells of the antigen plates,
incubated for 1 h at RT, washed 3 times with PBS-0.05% Tween 20,
and bound antibody detected by the addition of polyvalent
goat-anti-human IgG antibody conjugated with alkaline phosphatase
diluted 1:10.sup.3 with PBS-1% fetal calf serum. After the addition
of the substrate, 1 mg/ml p-nitrophenyl phosphate in carbonate
buffer (15 mM Na.sub.2CO.sub.3, 35 mM NaH.sub.2CO.sub.3, 10 mM
MgCl.sub.2 pH 9.6), the color development after 15 min was measured
in a EIA reader at 405 nm. "+" means OD405>1.0; "-" means
OD405<0.05. The negative control is <0.05. The results are
given in Table 1.
2TABLE 1 Reactivity of Antibody Fusion Proteins to Various Oral
Bacterial Strains Hitstatin Dhvar 5/SWLA3 1/SWLA3 Fusion Fusion
Oral Bacteria Strains Antibodies Antibodies S. mutans AATCC25175 +
+ LM7 + + OMZ175 + + S. Mitis ATCC49456 - - S. rattus ATCC19645 - -
S. sanguis ATCC49295 - - S sobrinus ATCC6715-B - - S. sobrinus
ATCC33478 - - L. acidophilus ATCC4356 - - L. casei ATCC11578 - - L.
plantarum ATCC14917 - - L. salivarius ATCC11742 - - A.
actinomycetemcomitans ATCC33384 - - A. naeslundi ATCC12104 - - A.
viscosus ATCC19246 - - Fusobacterium nucleatum ATCC25586 - -
Porphyromonas gingivalis ATCC33277 - -
[0112] The fusion proteins showed both specificity and
anti-microbial efficacy against S. mutans. Like the monoclonal
antibodies from which they are derived, the fusion proteins bind
specifically to S. mutans. (See Table 1). They also have
anti-bacterial efficacy against the bacteria, but are effective at
a much lower concentration than histatin 5 alone. (See Table
2).
3TABLE 2 Recombinant Histatin5/SWLA3 fusion antibodies targets S.
mutans with a great sensitivity and specificity Minimal Inhibitory
Concentrations S. mutans S. sanguis Host cells Histatin5 .about.10
.mu.M .about.10 .mu.M >50 .mu.M Histatin 5/SWLA3 .about.0.3
.mu.M .about.30 .mu.M >50 .mu.M fusion antibodies
[0113] This observation suggests that the recognition sequence is
responsible for specific binding between the fusion protein and S.
mutans, which locally enhances the concentration of histatin 5 at
the bacterial cell surface. At the concentration at which the
fusion protein showed antibacterial efficacy, the fusion proteins
showed no inhibitory effect on other bacteria or host cells (Table
2). Accordingly, these results suggest that the basic design
described herein may be useful for generating antibody-based fusion
proteins for treatment of other infections and infestations.
Example 2
Construction of a Chimeric Construct Containing Minibody and
Anti-Microbial Peptide, and its Expression in Yeast
[0114] a. Construction of SWLA3 Minibody-PG-1 Peptide Fusion
Protein
[0115] A minibody is a modified antibody molecule that comprises of
the variable regions of the heavy and light chain (V.sub.H and
V.sub.L) covalently linked via a short linker in a head-to-tail
fashion (see FIG. 5). To construct the SWLA3 minibody-PG-1
anti-microbial peptide fusion, PG-1 was linked to the N-terminus of
V.sub.H via a poly serine-glycine linker peptide, (SGGGG).sub.3
(SEQ ID NO. 17). The C-terminus of V.sub.H was connected via a
short GS linker, (GGGS).sub.2 (SEQ ID NO. 18), to the N-terminus of
V.sub.L.
[0116] The starting material for constructing the minibody was the
anti S. mutans monoclonal antibody, SWLA3, as described in the U.S.
Pat. No. 6,231,857. The anti-microbial peptide was protegrin PG-1
as described in the U.S. Pat. Nos. 5,693,486, 5,708,145, 5,804,558,
5,994,306, and 6,159,936 and Zhao et al., FEBS lett, 1994, 346
(2-3): 285-8.
[0117] The coding region of the protegrin PG-1 has the following
sequence: 5'-CGT GGC GGT CGC CTA TGC TAC TGT CGA CGT CGC TTT TGC
GTA TGC GTG GGA CGG TCT-3' (SEQ ID NO. 19). The gene was amplified
and fused with the linker region then to the V.sub.H region of
SWLA3 in the same fashion as described in Example 1 for Histadin5
fusion to SWLA3.
[0118] To construct PG-1-SWLA3 minibody, the PG-1-V.sub.H fragment
was amplified by PCR using primers PG-1F:
5'-GGGAATTCCGTGGCGGTCGCCTATGCTAC-3' (SEQ ID NO. 20), and VhR2:
5'-AGAGCCGCCACCCGAACCTCCGCCTGAAGAGACGGTGACTGAG- G-3' (SEQ ID NO.
21). The V.sub.L fragment was also amplified by PCR using primers
V.sub.LF2: 5'-GGTTCGGGTGGCGGCTCTGATGTTGTGATGACCCAGACT-3' (SEQ ID
NO. 22), and V.sub.LR2:
5'-GGTCTAGATTCCCAGAACCCCCACCCTTACGTTTCAGCTCCAGCTT- G-3' (SEQ ID NO.
23). The PG-1-V.sub.H and V.sub.L fragments were then joined by a
second PCR utilizing the complementarities between the 3' portion
of the PG-1-V.sub.H fragment and the 5' portion of the V.sub.L
fragment. The joined fragment was amplified by PCR using primers
PG-1F and V.sub.LR2. The fragment was subsequently cloned into a
cloning vector, pTOPO, and the correct sequence confirmed by DNA
sequencing.
[0119] b. Expression of PG-1-SWLA3 Minibody Fusion in the Yeast
Pichia pastoris.
[0120] Yeast Pichia pastoris is a commercially available system for
over expression of a large number of proteins including antibodies
and minibodies. To overexpress the PG-1-SWLA3 minibody fusion
protein in Pichia, the PG-1-V.sub.H-V.sub.L fragment was cloned
into the expression vector, pPICZ.alpha.A at the Eco RI-Xba I
sites. The cloning resulted in fusion of the N-terminus of PG-1
with the C-terminus of the .alpha.-factor signal sequence in the
vector, thus allowing secretion of the fusion protein to the
outside medium. The cloning also resulted in the fusion of the
C-terminus of V.sub.L to the N-terminus of the c-myc epitope and
the His-tag in the vector, allowing detection and purification of
the secreted protein. The construct was amplified in E. coli,
confirmed by sequencing, and transferred into Pichia. The correct
expression of the fusion protein was confirmed by Western blot
analysis, as well as by its ability to bind specifically to S.
mutans, the target bacteria of the original antibody SWLA3.
Example 3
Construction of Chimeric Construct Containing Surface-Binding
Peptide and Anti-Microbial Peptide
[0121] In addition to antibodies, some small peptide can also bind
to surface structures of microorganisms or eukaryotic cells. These
peptides, which we term "docking moiety", allow more flexibility
for the antimicrobial peptides (the killing moiety) to insert into
the cell membrane for killing. These peptides can be selected from
phage display libraries that contain random peptide sequences.
Phage-display libraries of 8-12 amino acids peptide are
commercially available. In this experiment, we have screened these
libraries for peptides capable of specifically binding to a target
organism, which can be bacteria, yeast, or other fungi. One or more
of these peptides will then be fused to the anti-microbial peptide
via a peptide linker, and expressed in an appropriate host, or
chemically synthesized.
[0122] a. Selection of Species-Specific Binding, Peptides with
Phase Display
[0123] We employed three phage display peptide libraries (New
England Biolabs) that contain >10.sup.9 unique
random-peptide-sequence-contain- ing phage clones. The Ph.D.-C7C
library displays 7-mer peptides with disulfide linkages, while the
Ph.D.-7 and Ph.D.-12 libraries contain completely randomized 7-mer
and 12-mer residues, respectively. The M13 filamentous phage used
for the procedure carried the random insert as an N-terminal fusion
to the minor coat protein pIII.
[0124] Briefly, 10.sup.10 pfu/ml of phage library was incubated
with 10.sup.9 bacterial cells for which targeting peptides are
desired. After centrifugation, unbound phage was removed by
aspiration. The pellet, which contained the target bacteria with
the bound phage, was washed several times using buffers containing
mild detergent to remove loosely bound phage particles, and the
tightly bound phage particles were eluted. This process is termed
panning.
[0125] The eluted phage was amplified by infecting E. coli F.sup.+
strains. After 3-4 rounds of panning and amplification, a phage
pool was obtained, which contained clones with high binding
affinity for the bacteria that it was panned against. Ten to twenty
phage clones from this pool were randomly picked for DNA
sequencing, from which the amino acid sequence of the peptide
insert was determined. By aligning the amino acid sequence of
multiple clones from the same phage pool, a consensus sequence for
the binding peptide was constructed. This consensus sequence
represents one of the binding peptides for this particular
bacterium.
[0126] To confirm the binding specificity of the consensus peptide,
the peptide was chemically synthesized and conjugated to FITC, a
green fluorescence dye. The labeled peptide was incubated with the
bacteria and analyzed by fluorescent microscopy for bacterial
species-specific binding. This methodology ensured that peptides
selected from phage display exhibits the same binding specificity
as a free peptide independent of the M13 phage particle.
[0127] a-1. Generation of Pseudomonas Specific Binding Peptides
Using Phage Display
[0128] Phage display technology was used as described above to
generate targeting peptides against Pseudomonas. After panning
against P. aeruginosa strain PAK cells, significant enrichment was
obtained with the PhD-12 library, compared to the other libraries,
after 3 rounds of panning (2 logs higher pfu/ml). A further
4.sup.th round of panning was carried out using more stringent
washing conditions to select for only the tightest binding clones,
and the titer of recovered bound phage for PhD-12
(4.5.times.10.sup.7 pfu/ml) remained high (Table 3). Ten clones
were then selected from the recovered PhD-12 library phage after
rounds 3 and 4. The sequences of the 3.sup.rd round clones are
shown in Table 4.
4TABLE 3 Panning of PhD-12, PhD-7 and PhD-C7C 1.sup.st Round
2.sup.nd Round 3.sup.rd Round 4.sup.th Round (pfu/ml) (pfu/ml)
(pfu/ml) (pfu/ml) Library input output input output input output
input output PhD-12 1 .times. 10.sup.10 4. .times. 10.sup.5 3
.times. 10.sup.10 3 .times. 10.sup.5 2 .times. 10.sup.10 2 .times.
10.sup.9 1 .times. 10.sup.10 4.5 .times. 10.sup.7 PhD-7 5 .times.
10.sup.10 3 .times. 10.sup.6 3 .times. 10.sup.10 2 .times. 10.sup.4
1 .times. 10.sup.10 2 .times. 10.sup.7 7 .times. 10.sup.9.sup. 1
.times. 10.sup.5 PhD-C7C 1 .times. 10.sup.10 1 .times. 10.sup.6 3
.times. 10.sup.10 3 .times. 10.sup.4 3 .times. 10.sup.10 6 .times.
10.sup.6 1 .times. 10.sup.11 1 .times. 10.sup.5
[0129]
5TABLE 4 Round 3 clones from PhD-12 PhD-12 clone 12:1 V P H E F K H
L Q M K P SEQ ID NO. 24 12:2 V P H E F K H L Q M K P SEQ ID NO. 25
12:3 H H H K A L A P T V T G SEQ ID NO. 26 12:4 V P H E F H A H R G
R L SEQ ID NO. 27 12:5 V P H E F K H L Q M K P SEQ ID NO. 28 12:6 Q
P H P H K V H S L P P SEQ ID NO. 29 12:7 V P H E F H A H R G R L
SEQ ID NO. 30 12:8 V P H E F H A H R G R L SEQ ID NO. 31 12:9 H H L
H Y N P A F P G L SEQ ID NO. 32 12:10 Q P A P Y I S S P S A S SEQ
ID NO. 33
[0130] Of the 3.sup.rd round sequences recovered, clones 12:1,
12:2, and 12:5 were identical, as well as clones 12:4, 12:7, and
12:8. These clones were probably originated from two clones. These
two original clones, though not identical, share sequences at the
N-terminal half of the peptide. Thus, a consensus sequence could be
deduced from this peptide pool.
[0131] a-2. Generation of Specific Binding Peptide Against
Staphylococcus aureus and E. coli from Phage Display Libraries.
[0132] Phage display technology was also used to generate peptides
specific for S. aureus and E. coli. The PhD-12 library was used for
the selections. The peptide sequences from the enriched clones are
summarized in Table 5 (against S. aureus) and Table 6 (against E.
coli).
6TABLE 5 Amino acid sequences of clones generated against S. aureus
SA5.1 V R L P L W L P S L N E SEQ ID NO. 34 SA5.3 A N Y F L P P V L
S S S SEQ ID NO. 35 SA5.4 S H P W N A Q R E L S V SEQ ID NO. 36
SA5.5 S V S V G M R P M P R P SEQ ID NO. 37 SA5.6 W T P L H P S T N
R P P SEQ ID NO. 38 SA5.7 S V S V G M K P S P R P SEQ ID NO. 39
SA5.8 S V S V G M K P S P R P SEQ ID NO. 40 SA5.9 S V P V G P Y N E
S Q P SEQ ID NO. 41 SA5.10 W A P P L F R S S L F Y SEQ ID NO. 42
SA2.2 W A P P X P X S S L F Y SEQ ID NO. 43 SA2.4 H H G W T H H W P
P P P SEQ ID NO. 44 SA2.5 S Y Y S L P P I F H I P SEQ ID NO. 45
SA2.6 H F Q E/N P L S R G G E L SEQ ID NO. 46 SA2.7 F S Y S P T R A
P L N M SEQ ID NO. 47 SA2.8 S X P X X M K X S X X X SEQ ID NO. 48
SA2.9 V S R H Q S W H P H D L SEQ ID NO. 49 SA2.10 D Y X Y R G L P
R X E T SEQ ID NO. 50 SA2.11 S V S V G M K P S P R P SEQ ID NO.
51
[0133]
7TABLE 6 Amino acid sequences of clones generated against E. coli
DH5.1 K H L Q N R S T G Y E T SEQ ID NO. 52 DH5.2 H I H S L S P S K
T W P SEQ ID NO. 53 DH5.3 T I T P T D A E M P F L SEQ ID NO. 54
DH5.4 H L L E S G V L E R G M SEQ ID NO. 55 DH5.5 H D R Y H I P P L
Q L H SEQ ID NO. 56 DH5.6 V N T L Q N V R H M A A SEQ ID NO. 57
DH5.7 S N Y M K L R A V S P F SEQ ID NO. 58 DH5.8 N L Q M P Y A W R
T E F SEQ ID NO. 59 DH5.9 Q K P L T G P H F S L I SEQ ID NO. 60
[0134] These results suggest that these species may have multiple
targets on the cell surface.
[0135] b. Rational Design of Targeting Peptides Based on
Biochemical/Biophysical Characteristics of Amino Acids and
Bacterial Surfaces
[0136] In addition to phage display, potential targeting peptides
can also be designed based on the biochemical and biophysical
characteristics of amino acids and the bacterial cell wall. For
example, positively charged peptide may bind to negatively charged
components on the cell surface and vice versa. Similarly,
hydrophobic peptides may bind to some hydrophobic pockets on the
cell surface based on hydrophobic interactions, or some peptide may
form a certain secondary structure that would fit into some
structures on the cell surface. By applying these principles, we
designed a panel of peptides and tested their binding potential
against a panel of Gram-positive and Gram-negative bacteria.
[0137] b-1. Design of Peptide
[0138] Peptide Cat-1 contained all cationic residues so that it
could potentially bind to negatively charged molecules on bacterial
surfaces. Phob-1 was made to be largely hydrophobic and attracted
to the hydrophobic cell wall constituents of Gram-positive
bacteria. LPTG-1 contained two "cell wall binding" repeats
separated by a flexible linker. These cell wall binding repeats are
specifically recognized by some streptococci and are involved in
bacterial adherence to surfaces. LPSB-1 and 2 consisted of repeats
of the LPS binding domains found in the sheep defensin SMAP-29.
Targeting peptide .alpha.-1 was designed to be 100%
.alpha.-helical, a characteristic common to many characterized
antimicrobial peptides and an important structural feature for
insertion into bacterial membranes. Anion-1 contained all
negatively charged amino acid residues while Philic-1 mainly
consisted of hydrophilic amino acid residues. Peptides were
synthesized by standard FastMoc chemistry.
[0139] b-1. Some representatives of designed targeting peptides
8 Cat-1 LPSB-1 KKHRKHRKHRKH RGLRRLGRRGLRRLGR 12 aa (SEQ ID NO. 61)
16 aa (SEQ ID NO. 62) Phob-1 LPSB-2 KPVLPVLPVLPVL VLRIIRIAVLRIIRIA
12 aa (SEQ ID NO. 63) 16 aa (SEQ ID NO. 64) LPTG-1 .alpha.-1
LPETGGSGGSLPETG RAHIRRAHIRR 17 aa (SEQ ID NO. 65) 11 aa (SEQ ID NO.
66) Anion-1 Philic-1 DEDEDDEEDDDEEE STMCGSTMCGSTMCG 14 aa (SEQ ID
NO. 67) 15 aa (SEQ ID NO. 68)
[0140] b-2. Testing Binding Activity of the Designed Targeting
Peptides
[0141] Presented here are some examples for testing the specificity
of the designed targeting peptides. The peptides were labeled at
the N-terminus with the fluorescent dye FITC, and the labeled
peptides were allowed to bind to the bacteria as described
previously. Fluorescent microscopy was then used to determine the
binding efficiency based on the brightness of the bacterial cells
after peptide binding.
9TABLE 7 Specificity of targeting peptides against log phase cells
Bacterial strains LPTG-1 Cat-1 Phob-1 Dye alone E. coli Aw405 - + -
- E. coli DH5.alpha. - ++ - - E. coli ER2738 - ++ - - E. coli W3110
+ ++ - - E. coli Mg1655 - + - - P. aeruginosa Pak - ++++ - - P.
mendocina - ++++ - - S. mutans - - - - S. epidermidis + - - - S.
aureus + - - - T. denticola - - - - M. xanthus - - - -
[0142] Data presented in Table 7 clearly show that Cat-1 binds
specifically to Pseudomonas and E. coli with a much stronger
binding to the former species. In contrast, Phob-1 did not bind to
any of the bacterial species that we tested. LPTG-1, on the other
hand, showed weak binding to the two staphylococcal species.
[0143] To further confirm the specificity of Cat-1 binding to Pak
cells, competition-binding experiments were conducted. Labeled
Cat-1 was mixed with increasing concentrations of unlabeled Cat-1,
and fluorescent labeling of the target bacterial cells was measured
by fluorescent microscopy. The results demonstrated that
fluorescent signal of labeled Cat-1 on PAK cells was reduced upon
addition of equal molar concentration of unlabeled Cat-1, and
completely abolished at 10.times.molar excess unlabeled Cat-1.
[0144] c. Construction of a Species-Specific Antimicrobial Peptide
with High Specificity, Sensitivity, and Much Improved Killing
Kinetics.
[0145] With the successful design of a targeting peptide (CAT-1)
that can specifically bind to Pseudomonas and to E. coli to a
lesser degree, one can fuse this targeting peptide to a
non-specific antimicrobial peptide, making it a species-specific
killing peptide. Below is an example for constructing such a
species-specific antimicrobial peptide and testing its specificity
and killing efficiency against its targeting bacteria. This example
demonstrates that this novel technology can be used to develop
targeted antimicrobial therapy against other pathogenic bacteria,
fungi, and viruses.
[0146] c-1. Design of the Fusion Peptides
[0147] Novispirin G10 is a wide-spectrum antimicrobial peptide,
which is active against both Gram-positive and Gram-negative
bacteria. Two forms of the fusion peptides were constructed.
G10CatC contained the targeting peptide Cat-1 domain fused at its
C-terminus to G10 through a five-residue linker, GGSGG (SEQ ID NO.
69), to allow flexibility of the targeting and killing domains.
G10CatN had an opposite configuration as G10CatC in that the
killing domain G10 was at the N-terminal portion of the fusion
peptide. The sequences of the two fusion peptides are shown
below.
10 G10CatC KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYG (SEQ ID NO. 70)
Cat-1 domain G10 domain G10CatN
KNLRRIIRKGIHIIKKYGGGSGGSKKHRKHRKHRKH (SEQ ID NO. 71) G10 domain
Cat-1 domain
[0148] c-2. Determination of the Minimum Bactericidal Concentration
(MBC) of the Fusion Peptides
[0149] To test the antimicrobial activity of the fusion peptides
against target bacteria, a panel of bacteria, both gram positive
and gram negative, were tested. The results of the minimum
bactericidal concentration (MBC) are summarized in Table 8.
11TABLE 8 MBCs of G10, G10CatC and G10CatN against a selected panel
of bacteria MBC (.mu.M) Bacteria G10 G10C G10N Salmonella Lt2
(ATCC) >22.5 11.6-23.2 >23.2 Salmonella Lt7 >22.5 >23.2
>23.2 E. coli 3132 (wt) 11.25 5.8 11.6 E. coli W3110 1.4 2.81
>11.6 E. coli W3110 ampR 2.8 2.9 >23.2 E. coli Aw405 2.8 11.6
>23.2 E. coli Mg1665 (smooth) 2.8 2.9 >23.2 P. mendocina 1.05
1.4-2.4 2.8 P. aeruginosa Pak (ATCC) 42.18 4.9 17.4 P. aeruginosa
Pak 54.5 1.4-2.9 45 P. fluorescens 1088 1.4 1.45 1.45 P.
fluorescens 1089 >11.25 2.9 11.6 P. tabaci 5.625 2.175 >11.6
B. cepacia 23 13 11.6 Y. entercolitica 22.5 23.2 >23.2
Myxococcus >11.25 >11.6 >11.6 Klebsiella Kay2026 >11.25
11.6-23.2 >11.6 S. aureus >22.5 >23.2 >23.2 S. mutans
140 3.5-7.03 3.6-7.25 ND S. mutans 159 3.5-7.03 3.6-7.25 ND S.
sanguis 3.5-7.03 3.6-7.25 ND S. oralis >58 >58 ND S. mitis
>58 >58 ND
[0150] In general, G10 and G10CatC are more active against E. coli,
Pseudomonas, and mutans streptococci, and less active against
Salmonella, Staphylococcus, Yersinia, and the non-mutans
streptococci. Of particular notice is that fusion of the targeting
peptide to the N-terminus of G10 (G10CatC) enhanced killing
activity 38-fold against Pak, but decreased killing activity 2-fold
against P. mendocina, while the same fusion did not change the
killing activity against S. mutans. In contrast, fusion of the
targeting peptide to the C-terminus of G10 (G10CatN) significantly
reduced killing activity against E. coli, but did not change
killing activity against others. These results indicate that the
position of the targeting peptide relative to the killing peptide
plays an important role in determining the killing activity of the
fusion peptide. Note that the targeting peptide CAT-1 alone does
not have any antimicrobial activity.
[0151] c-3. Determination of Killing Kinetics
[0152] For non-specific antimicrobial peptide, the number of
molecules bound to the cell surface is proportional to the peptide
concentration in the solution, because binding is solely dependent
on passive diffusion of the peptide through solution. However, if a
targeting peptide is fused to the killing peptide, a higher
concentration of peptide on the target cell surface could be
achieved in a short period of time due to the high affinity, active
binding of the targeting peptide to the target bacterial cells.
[0153] To show that increased killing activity of the fusion
peptide is due to enhanced binding to the cell surface, time-kill
assays were performed using a selected number of species, i.e. P.
aeruginosa, and P. mendocina. Briefly, varying concentrations of
peptide G10, G10CatC, or G10CatN was added to 10.sup.5 of target
bacterial cells and survivors were counted by plating samples after
0.5, 1, 2 and 4 minutes of peptide exposure. Results are summarized
in FIGS. 6-10.
[0154] Strikingly, at 9 .mu.M concentration, the fusion peptide
G10CatC achieved 2.5-log killing after 30 seconds, while treatment
with G10 alone did not show significant killing until 4 minutes (1
log.sub.10 reduction). Concurrently, at 3 .mu.M concentration,
G10CatC treatment reduced the number of live P. mendocina cells to
below detectable levels within 4 min, in contrast, G10 alone did
not display any killing activity at the same concentration during
the same time period. These data suggest that the addition of a
targeting domain significantly improves the short-term killing rate
against the target bacteria compared with the killing peptide
alone. In contrast to G10C, G10CatN displayed no killing at any
concentration tested through 4 minutes, suggesting that the G10
peptide must be fused to the C-terminus of the targeting domain to
facilitate killing activity.
[0155] Of particular notice for P. mendocina is that although
G10CatC has a 2.times. higher MBC than G10 alone, it kills much
quicker in the kinetic analysis. This indicates that specific
binding to the target cell surface allowed local accumulation of
high concentration-of the peptide in a short period of time (<4
minutes), thus enhanced killing kinetics.
[0156] The killing kinetics for PAK cells is similar (FIGS. 9 and
10). Here, G10 alone did not show any killing activity even at 18
.mu.M concentration, in contrast, G10CatC killed >90% of PAK
cells within 4 min at a lower concentration (9 .mu.M).
[0157] Since G10 alone did not show significant killing activity
within 4 min, another time-kill experiment was conducted with
treatments lasting up to 2 h to get a more complete picture of
killing kinetics. The results are summarized in FIG. 11. Similar to
the short-term kinetic studies described above, treatment with 3
.mu.M G10CatC killed all input P. mendocina cells within 30 min, in
contrast, it took 2 h for G10 alone to kill all input P. mendocina
cells at the same concentration. G10CatC also showed a more
pronounced enhanced killing over G10 at 9 .mu.M; no surviving P.
mendocina cells were recovered after 2 minutes of treatment with
G10CatC, but a significant number of cells were recovered even
after 30 minutes of G10 exposure.
[0158] c-4. Determination of Killing Specificity
[0159] To test if G10CatC can selectively kill its target bacteria
in a mixed culture, 10.sup.5 of P. mendocina and E. coli W3110
cells were mixed and treated with 6 .mu.M G10 or G10CatC peptides.
Samples were taken at 30 sec. 1 min, 2 min, and 4 min, and
survivors were counted by plating assays. Preliminary results are
summarized in FIG. 12 and 13. It is apparent that treatment with
G10CatC selectively reduced the number of P. mendocina cells from
the mixed culture, and by 4 min no live P. mendocina cells could be
detected. In contrast, the number of live W3110 cells remained
nearly unchanged during the same period of treatment. As expected,
treatment with G10 alone for 4 min did not affect the survival of
P. mendocina nor W3110 cells.
[0160] To further demonstrate this effect, another mixed culture
killing experiment was performed, in which PAK cells were mixed
with S. mutans cells, and the cell mixture was treated with 9 .mu.M
G10CatC or G10 alone. As shown in FIGS. 14 and 15, G10 alone did
not kill any of the cells within 4 min. In contrast, G10CatC killed
over 90% of PAK cells, but none of S. mutans cells.
[0161] Taken together, these results demonstrate that G10CatC can
specifically kill the bacterial species to which it specifically
binds (the target cells), but not the non-target cells, although
both types of cells are equally sensitive to the killing activity
of the peptide (as demonstrated in MBC assay).
[0162] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
71 1 563 DNA Artificial sequence Synthesized using sequential PCR
techniques 1 ggatatccac catggacttc gggttgagct tggttttcct tgtccttact
ttaaaaggtg 60 tccagtgtga tagccacgct aagcggcacc acggatataa
gcggaagttc cacgagaagc 120 accactcgca cagaggatac tctggtggcg
gtggctcggg cggaggtggg tcgggtggcg 180 gcggatccga cgtgaagctt
gtggagtctg ggggaggctt agtgaaccct ggagggtccc 240 tgaaactctc
ctgtgcagcc tctggattca ctttcagtag ctataccatg tcttgggttc 300
gccagactcc ggagaagagg ctggagtggg tcgcatccat tagtagtggt ggtacttaca
360 cctactatcc agacagtgtg aagggccgat tcaccatctc cagagacaat
gccaagaaca 420 ccctgtacct gcaaatgacc agtctgaagt ctgaggacac
agccatgtat tactgttcaa 480 gagatgacgg ctcctacggc tcctattact
atgctatgga ctactggggt caaggaacct 540 cagtcaccgt ctcttcagct agc 563
2 165 PRT Artificial sequence Synthesized using sequential PCR
techniques 2 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys
Phe His Glu 1 5 10 15 Lys His His Ser His Arg Gly Tyr Ser Gly Gly
Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Asp
Val Lys Leu Val Glu Ser Gly 35 40 45 Gly Gly Leu Val Asn Pro Gly
Gly Ser Leu Lys Leu Ser Cys Ala Ala 50 55 60 Ser Gly Phe Thr Phe
Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr 65 70 75 80 Pro Glu Lys
Arg Leu Glu Trp Val Ala Ser Ile Ser Ser Gly Gly Thr 85 90 95 Tyr
Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 100 105
110 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Thr Ser Leu Lys Ser
115 120 125 Glu Asp Thr Ala Met Tyr Tyr Cys Ser Arg Asp Asp Gly Ser
Tyr Gly 130 135 140 Ser Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr 145 150 155 160 Val Ser Ser Ala Ser 165 3 533 DNA
Artificial sequence Synthesized using sequential PCR techniques 3
ggatatccac catggacttc gggttgagct tggttttcct tgtccttact ttaaaaggtg
60 tccagtgtaa gcggctgttt aaggagctca agttcagcct gcgcaagtac
tctggtggcg 120 gtggctcggg cggaggtggg tcgggtggcg gcggatccga
cgtgaagctt gtggagtctg 180 ggggaggctt agtgaaccct ggagggtccc
tgaaactctc ctgtgcagcc tctggattca 240 ctttcagtag ctataccatg
tcttgggttc gccagactcc ggagaagagg ctggagtggg 300 tcgcatccat
tagtagtggt ggtacttaca cctactatcc agacagtgtg aagggccgat 360
tcaccatctc cagagacaat gccaagaaca ccctgtacct gcaaatgacc agtctgaagt
420 ctgaggacac agccatgtat tactgttcaa gagatgacgg ctcctacggc
tcctattact 480 atgctatgga ctactggggt caaggaacct cagtcaccgt
ctcttcagct agc 533 4 155 PRT Artificial sequence Synthesized using
sequential PCR techniques 4 Lys Arg Leu Phe Lys Glu Leu Lys Phe Ser
Leu Arg Lys Tyr Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Val 20 25 30 Lys Leu Val Glu Ser Gly
Gly Gly Leu Val Asn Pro Gly Gly Ser Leu 35 40 45 Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met 50 55 60 Ser Trp
Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser 65 70 75 80
Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly 85
90 95 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
Gln 100 105 110 Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys Ser Arg 115 120 125 Asp Asp Gly Ser Tyr Gly Ser Tyr Tyr Tyr Ala
Met Asp Tyr Trp Gly 130 135 140 Gln Gly Thr Ser Val Thr Val Ser Ser
Ala Ser 145 150 155 5 24 PRT Artificial sequence Synthesized using
sequential PCR techniques 5 Asp Ser His Ala Lys Arg His His Gly Tyr
Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg Gly Tyr
20 6 14 PRT Artificial sequence Synthesized using sequential PCR
techniques 6 Lys Arg Leu Phe Lys Glu Leu Lys Phe Ser Leu Arg Lys
Tyr 1 5 10 7 89 DNA Artificial sequence Primer 986 7 caccactcgc
acagaggata ctctggtggc ggtggctcgg gcggaggtgg gtcgggtggc 60
ggcggatccg acgtgaagct tgtggagtc 89 8 84 DNA Artificial sequence
Primer 987 8 ggtgtccagt gtgatagcca cgctaagcgg caccacggat ataagcggaa
gttccacgag 60 aagcaccact cgcacagagg atac 84 9 74 DNA Artificial
sequence Primer 988 9 gatatccacc atggacttcg ggttgagctt ggttttcctt
gtccttactt taaaaggtgt 60 ccagtgtgat agcc 74 10 87 DNA Artificial
sequence Primer 989 10 gttcagcctg cgcaagtact ctggtggcgg tggctcgggc
ggaggtgggt cgggtggcgg 60 cggatccgac gtgaagcttg tggagtc 87 11 69 DNA
Artificial sequence Primer 990 11 gtccttactt taaaaggtgt ccagtgtaag
cggctgttta aggagctcaa gttcagcctg 60 cgcaagtac 69 12 65 DNA
Artificial sequence Primer 991 12 ggatatccac catggacttc gggttgagct
tggttttcct tgtccttact ttaaaaggtg 60 tccag 65 13 39 DNA Artificial
sequence Primer 452 13 tgggtcgacw gatggggstg ttgtgctagc tgaggagac
39 14 12 PRT Artificial sequence Consensus sequence 14 Xaa Xaa His
Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 18 PRT Artificial
sequence Protegrin PG-1 15 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg
Arg Phe Cys Val Cys Val 1 5 10 15 Gly Arg 16 18 PRT Artificial
sequence Novispirin G10 16 Lys Asn Leu Arg Arg Ile Ile Arg Lys Gly
Ile His Ile Ile Lys Lys 1 5 10 15 Tyr Gly 17 15 PRT Artificial
sequence Linker peptide 17 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly 1 5 10 15 18 8 PRT Artificial sequence Linker
peptide 18 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 19 57 DNA Artificial
sequence Anti-microbial peptide 19 cgtggcggtc gcctatgcta ctgtcgacgt
cgcttttgcg tatgcgtggg acggtct 57 20 29 DNA Artificial sequence
Amplification primer 20 gggaattccg tggcggtcgc ctatgctac 29 21 44
DNA Artificial sequence Amplification primer 21 agagccgcca
cccgaacctc cgcctgaaga gacggtgact gagg 44 22 39 DNA Artificial
sequence Amplification primer 22 ggttcgggtg gcggctctga tgttgtgatg
acccagact 39 23 47 DNA Artificial sequence Amplification primer 23
ggtctagatt cccagaaccc ccacccttac gtttcagctc cagcttg 47 24 12 PRT
Artificial sequence Peptide library 24 Val Pro His Glu Phe Lys His
Leu Gln Met Lys Pro 1 5 10 25 12 PRT Artificial sequence Peptide
library 25 Val Pro His Glu Phe Lys His Leu Gln Met Lys Pro 1 5 10
26 12 PRT Artificial sequence Peptide library 26 His His His Lys
Ala Leu Ala Pro Thr Val Thr Gly 1 5 10 27 12 PRT Artificial
sequence Peptide library 27 Val Pro His Glu Phe His Ala His Arg Gly
Arg Leu 1 5 10 28 12 PRT Artificial sequence Peptide library 28 Val
Pro His Glu Phe Lys His Leu Gln Met Lys Pro 1 5 10 29 12 PRT
Artificial sequence Peptide library 29 Gln Pro His Pro His Lys Val
His Ser Leu Pro Pro 1 5 10 30 12 PRT Artificial sequence Peptide
library 30 Val Pro His Glu Phe His Ala His Arg Gly Arg Leu 1 5 10
31 12 PRT Artificial sequence Peptide library 31 Val Pro His Glu
Phe His Ala His Arg Gly Arg Leu 1 5 10 32 12 PRT Artificial
sequence Peptide library 32 His His Leu His Tyr Asn Pro Ala Phe Pro
Gly Leu 1 5 10 33 12 PRT Artificial sequence Peptide library 33 Gln
Pro Ala Pro Tyr Ile Ser Ser Pro Ser Ala Ser 1 5 10 34 12 PRT
Artificial sequence Peptide library 34 Val Arg Leu Pro Leu Trp Leu
Pro Ser Leu Asn Glu 1 5 10 35 12 PRT Artificial sequence Peptide
library 35 Ala Asn Tyr Phe Leu Pro Pro Val Leu Ser Ser Ser 1 5 10
36 12 PRT Artificial sequence Peptide library 36 Ser His Pro Trp
Asn Ala Gln Arg Glu Leu Ser Val 1 5 10 37 12 PRT Artificial
sequence Peptide library 37 Ser Val Ser Val Gly Met Arg Pro Met Pro
Arg Pro 1 5 10 38 12 PRT Artificial sequence Peptide library 38 Trp
Thr Pro Leu His Pro Ser Thr Asn Arg Pro Pro 1 5 10 39 12 PRT
Artificial sequence Peptide library 39 Ser Val Ser Val Gly Met Lys
Pro Ser Pro Arg Pro 1 5 10 40 12 PRT Artificial sequence Peptide
library 40 Ser Val Ser Val Gly Met Lys Pro Ser Pro Arg Pro 1 5 10
41 12 PRT Artificial sequence Peptide library 41 Ser Val Pro Val
Gly Pro Tyr Asn Glu Ser Gln Pro 1 5 10 42 12 PRT Artificial
sequence Peptide library 42 Trp Ala Pro Pro Leu Phe Arg Ser Ser Leu
Phe Tyr 1 5 10 43 12 PRT Artificial sequence Peptide library 43 Trp
Ala Pro Pro Xaa Pro Xaa Ser Ser Leu Phe Tyr 1 5 10 44 12 PRT
Artificial sequence Peptide library 44 His His Gly Trp Thr His His
Trp Pro Pro Pro Pro 1 5 10 45 12 PRT Artificial sequence Peptide
library 45 Ser Tyr Tyr Ser Leu Pro Pro Ile Phe His Ile Pro 1 5 10
46 13 PRT Artificial sequence Peptide library 46 His Phe Gln Glu
Asn Pro Leu Ser Arg Gly Gly Glu Leu 1 5 10 47 12 PRT Artificial
sequence Peptide library 47 Phe Ser Tyr Ser Pro Thr Arg Ala Pro Leu
Asn Met 1 5 10 48 12 PRT Artificial sequence Peptide library 48 Ser
Xaa Pro Xaa Xaa Met Lys Xaa Ser Xaa Xaa Xaa 1 5 10 49 12 PRT
Artificial sequence Peptide library 49 Val Ser Arg His Gln Ser Trp
His Pro His Asp Leu 1 5 10 50 12 PRT Artificial sequence Peptide
library 50 Asp Tyr Xaa Tyr Arg Gly Leu Pro Arg Xaa Glu Thr 1 5 10
51 12 PRT Artificial sequence Peptide library 51 Ser Val Ser Val
Gly Met Lys Pro Ser Pro Arg Pro 1 5 10 52 12 PRT Artificial
sequence Peptide library 52 Lys His Leu Gln Asn Arg Ser Thr Gly Tyr
Glu Thr 1 5 10 53 12 PRT Artificial sequence Peptide library 53 His
Ile His Ser Leu Ser Pro Ser Lys Thr Trp Pro 1 5 10 54 12 PRT
Artificial sequence Peptide library 54 Thr Ile Thr Pro Thr Asp Ala
Glu Met Pro Phe Leu 1 5 10 55 12 PRT Artificial sequence Peptide
library 55 His Leu Leu Glu Ser Gly Val Leu Glu Arg Gly Met 1 5 10
56 12 PRT Artificial sequence Peptide library 56 His Asp Arg Tyr
His Ile Pro Pro Leu Gln Leu His 1 5 10 57 12 PRT Artificial
sequence Peptide library 57 Val Asn Thr Leu Gln Asn Val Arg His Met
Ala Ala 1 5 10 58 12 PRT Artificial sequence Peptide library 58 Ser
Asn Tyr Met Lys Leu Arg Ala Val Ser Pro Phe 1 5 10 59 12 PRT
Artificial sequence Peptide library 59 Asn Leu Gln Met Pro Tyr Ala
Trp Arg Thr Glu Phe 1 5 10 60 12 PRT Artificial sequence Peptide
library 60 Gln Lys Pro Leu Thr Gly Pro His Phe Ser Leu Ile 1 5 10
61 12 PRT Artificial sequence Design peptide 61 Lys Lys His Arg Lys
His Arg Lys His Arg Lys His 1 5 10 62 16 PRT Artificial sequence
Design peptide 62 Arg Gly Leu Arg Arg Leu Gly Arg Arg Gly Leu Arg
Arg Leu Gly Arg 1 5 10 15 63 13 PRT Artificial sequence Design
peptide 63 Lys Pro Val Leu Pro Val Leu Pro Val Leu Pro Val Leu 1 5
10 64 16 PRT Artificial sequence Design peptide 64 Val Leu Arg Ile
Ile Arg Ile Ala Val Leu Arg Ile Ile Arg Ile Ala 1 5 10 15 65 15 PRT
Artificial sequence Design peptide 65 Leu Pro Glu Thr Gly Gly Ser
Gly Gly Ser Leu Pro Glu Thr Gly 1 5 10 15 66 11 PRT Artificial
sequence Design peptide 66 Arg Ala His Ile Arg Arg Ala His Ile Arg
Arg 1 5 10 67 14 PRT Artificial sequence Design peptide 67 Asp Glu
Asp Glu Asp Asp Glu Glu Asp Asp Asp Glu Glu Glu 1 5 10 68 15 PRT
Artificial sequence Design peptide 68 Ser Thr Met Cys Gly Ser Thr
Met Cys Gly Ser Thr Met Cys Gly 1 5 10 15 69 5 PRT Artificial
sequence Linker peptide 69 Gly Gly Ser Gly Gly 1 5 70 36 PRT
Artificial sequence Fusion peptide 70 Lys Lys His Arg Lys His Arg
Lys His Arg Lys His Gly Gly Ser Gly 1 5 10 15 Gly Ser Lys Asn Leu
Arg Arg Ile Ile Arg Lys Gly Ile His Ile Ile 20 25 30 Lys Lys Tyr
Gly 35 71 36 PRT Artificial sequence Fusion peptide 71 Lys Asn Leu
Arg Arg Ile Ile Arg Lys Gly Ile His Ile Ile Lys Lys 1 5 10 15 Tyr
Gly Gly Gly Ser Gly Gly Ser Lys Lys His Arg Lys His Arg Lys 20 25
30 His Arg Lys His 35
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